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Abstract

Each eukaryotic cell stores a tremendous amount of DNA (measuring about two meters in length) within its microscopic nucleus. To achieve this unlikely feat, eukaryotes compact their DNA into a dense, proteinaceous structure called chromatin. However, chromatin is generally inhibitory to essential processes that require access to DNA, such as transcription, DNA replication and recombination. Therefore, cells require active mechanisms to regulate chromatin structure. The i̱mitation switch (ISWI) family of ATP-dependent chromatin remodeling factors are conserved throughout eukaryotes and utilize the energy of ATP to mobilize nucleosomes (the fundamental units of chromatin) on DNA. Members of the ISWI family have been shown to catalyze nucleosome sliding, spacing and loading activities in vitro. However, the in vivo functions of ISWI complexes, and the mechanisms by which they remodel chromatin in vivo, remain largely unknown. To gain some insight into these questions, I have undertaken an analysis of the Isw2 complex, using yeast as a model system. I found that Isw2 complex functions to repress transcription of early meiotic genes in a parallel pathway to the Sin3-Rpd3 histone deacetylase (HDAC) complex in vivo. This was the first example of two mechanistically different chromatin remodeling complexes functioning in parallel to repress transcription. Using whole genome expression analysis, I found that the parallel pathways of repression by the Sin3-Rpd3 and Isw2 complexes extends to a substantial number of yeast genes in many functional categories. By mapping the positions of nucleosomes in the presence or absence of Isw2 complex, I found that Isw2 complex represses transcription by creating compact, nuclease-inaccessible chromatin structure near the promoters of target genes. To understand the mechanism by which Isw2 complex creates this compact chromatin structure, I created an inducible allele of the ISW2 gene. Upon induction, I found that Isw2 complex slides nucleosomes unidirectionally toward the promoters of two target genes, reducing the spacing between nucleosomes as a result. This was the first demonstration of nucleosome sliding in vivo by an ATP-dependent chromatin remodeling factor, and has set the stage for experiments to probe the molecular details of nucleosome sliding by ISWI complexes at their physiological targets.